CN117700552B

CN117700552B - Anti-human CD93 protein rabbit monoclonal antibody and application thereof

Info

Publication number: CN117700552B
Application number: CN202311708003.6A
Authority: CN
Inventors: 邹彪; 吴海; 孙倩; 郭夏阳; 黄谧; 王雪
Original assignee: Wuhan Abclonal Inc
Current assignee: Wuhan Abclonal Inc
Priority date: 2023-12-11
Filing date: 2023-12-11
Publication date: 2024-08-06
Anticipated expiration: 2043-12-11
Also published as: CN117700552A

Abstract

The invention belongs to the technical field of antibody preparation, and particularly relates to a rabbit monoclonal antibody for resisting human CD93 protein and application thereof. The amino acid sequences of the complementarity determining regions 1-3 on the light chain variable region of the rabbit monoclonal antibody are respectively shown as SEQ ID NO.3-5, and the amino acid sequences of the complementarity determining regions 1-3 on the heavy chain variable region are respectively shown as SEQ ID NO. 8-10. The invention prepares the rabbit monoclonal antibody by taking the extracellular region of the CD93 protein as immunogen, the antibody can specifically identify the recombinant expressed CD93 protein and the cell expressed natural CD93 protein, and has high affinity, good specificity and strong anti-interference capability, can avoid false positive or false negative results caused by nonspecific binding and the like during immunodetection, is favorable for improving the accuracy and reliability of immunodetection, especially the analysis result of flow cell, and is suitable for detecting the CD93 protein with high specificity and high sensitivity.

Description

Anti-human CD93 protein rabbit monoclonal antibody and application thereof

Technical Field

The invention relates to the technical field of antibody preparation, in particular to a rabbit monoclonal antibody for resisting human CD93 protein and application thereof.

Background

Melanoma cell adhesion molecule CD93, also known as C1qR1, C1qR, C1qRP, MXRA4, ECSM3, is a type I single transmembrane glycoprotein consisting of a C-type lectin domain, an epidermal growth factor (EPIDERMAL GROWTH FACTOR, EGF) -like domain, a highly glycosylated mucin-like domain, which is a transmembrane protein, and can be divided into an extracellular domain (amino acid sequence at positions 24-580), a transmembrane domain (amino acid sequence at positions 581-601) and a short cytoplasmic domain (amino acid sequence at positions 601-652). CD93 is expressed mainly in endothelial cells, platelets, stem cells and bone marrow cells (granulocytes and monocytes), and is involved in various physiological processes such as angiogenesis, cytoskeletal dynamics, migration and adhesion of endothelial cells, apoptosis and remodeling of extracellular matrix (ExtraCellular Matrix, ECM), etc.

Early in the study, researchers thought CD93 was an immune molecule involved in the adhesion and penetration of immune cells, and was associated with various inflammatory and immune-related diseases. In 2020, researchers at the university of medical college of extension of korea have found that allergic asthma is associated with significant changes in CD93 expression and serum levels in vitro and in vivo models, and allergen House Dust Mite (HDM) stimulation causes the extracellular domain of CD93 expressed in bronchial epithelial cells to shed into serum in a soluble form, thus CD93 can be detected in serum under allergic stimulation, revealing a potential role for CD93 in predicting human asthma. Recent literature reports that CD93 is a novel angiogenesis activator, involved in proliferation, migration and neovascularization of endothelial cells, highly expressed in endothelial cells of tumor vessels and under expressed in non-proliferating endothelial cells, and its high expression accelerates tumor growth and reduces host survival. Angiogenesis is a key step in the development of malignant tumors, tumor blood vessels are the trophic pathways and metastasis pathways of solid tumor cells, and when tumor volumes are greater than 2mm, vascular endothelial growth factor (Vascular Endothelial Growth Factor, VEGF) begins to secrete, grow rapidly and provide a metastasis pathway. In 2021, the university of yarrowia show flat subject group and the university of corradon Anschuz division Zhu Yuwen subject group have published a study titled "Blockade of the CD93 pathway normalizes tumor vasculature to facilitate drug delivery and immunotherapy" in journal Science Translational Medicine, and by comparing the analysis of gene expression profiles of tumors under in vivo treatment with VEGF inhibitors, CD93 was found to be a candidate receptor for down-regulation in VEGF inhibition and also a potential target for mediating vascular normalization, and the pro-angiogenic effect of CD93 in endothelial cells was also demonstrated. in addition, CD93 was found to be an important regulator of self-renewal and proliferation of mouse and human Leukemia stem cells (Leukemia STEM CELLS, LSCs), rather than hematopoietic stem cells, which are leukemic initiating cells in acute myeloid Leukemia (acute myeloid Leukemia, AML), chronic myeloid Leukemia (chronic myeloid Leukemia, CML) and other hematological tumors, and self-renewal of LSCs is a key feature in the development and maintenance of Leukemia. The intracellular domain of CD93 is independent of the extracellular domain connection, and can promote gene transcription through a transcription regulator SCY 1-like pseudo kinase 1, and can be used as an important regulator and a potential therapeutic target of CML LSCs. Previously, CD93 has been demonstrated to be a marker signal for CML LSCs that persist after TKI treatment, inducing proliferation and disease progression of CML LSCs carrying a mixed line leukemia (mixed lineage leukemia, MLL) gene rearrangement. It can be seen that the potential of CD93 in hematological neoplasms is also not insignificant.

Previous studies have demonstrated a critical role for CD93 in malignant tumors such as solid tumors and hemangiomas, and up-regulation of CD93 in tumor vessels as a biomarker for certain diseases, over-expression of CD93 in tumor vessels can be observed in pancreatic tumors, human kidney cancer, head and neck cancer, colon cancer, and the like. Early identification of CD93 expression in tumor tissue is therefore critical for patient diagnosis and treatment options for the type of clinical pathology. In recent years, along with the rapid development of immunological research, an immunological detection technology based on antigen-antibody specific reaction is highly valued, and the detection of CD93 expression condition by specific antibody targeting CD93 has good application prospect. However, most of the related anti-human CD93 antibodies in the prior art are murine monoclonal antibodies (such as antibodies produced by Biolegend, thermo FISHER SCIENTIFIC and the like) or goat polyclonal antibodies (such as antibodies produced by R & D Systems), and these antibodies have low specificity and are difficult to use in the field of immunological detection, especially in the field of flow cytometry. In the flow cytometry detection process, the content of target antigen is low, interference factors are more, and the low specificity of the antibody easily causes nonspecific binding, so that the accuracy or sensitivity of a detection result is influenced. Therefore, the development of the high-specificity antibody capable of meeting the application scene of flow cytometry analysis has important significance for high-sensitivity detection of human CD93 protein.

Disclosure of Invention

Aiming at the problems that the anti-human CD93 protein antibody in the prior art has poor specificity and is difficult to be used for flow cell detection and the like, the invention provides a rabbit monoclonal antibody for resisting human CD93 protein and provides application of the rabbit monoclonal antibody in preparation of a human CD93 protein immunoassay kit.

In order to achieve the above purpose, the present invention is specifically realized by the following technical scheme:

The first aspect of the invention provides a rabbit monoclonal antibody against human CD93 protein, comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of a complementarity determining region 1, a complementarity determining region 2 and a complementarity determining region 3 on the light chain variable region are shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively; the amino acid sequences of the complementarity determining regions 1, 2 and 3 on the heavy chain variable region are shown in SEQ ID NO.8, 9 and 10, respectively.

Further, the amino acid sequence of the light chain variable region is shown as SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 7.

Further, the light chain constant region and the heavy chain constant region form a light chain, and the amino acid sequence of the light chain is shown as SEQ ID NO. 1; the heavy chain constant region and the heavy chain variable region form a heavy chain, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 6.

Further, the rabbit monoclonal antibody is a full-length antibody or an antibody fragment having immunological activity; the antibody fragment is selected from at least one of Fab fragment, F (ab) ₂ fragment, fv fragment, (Fv) ₂ fragment, scFv fragment and sc (Fv) ₂ fragment.

Further, the immunogen for preparing the rabbit monoclonal antibody is CD93 protein 24-580aa.

In a second aspect the invention provides a nucleic acid molecule encoding a rabbit monoclonal antibody as described above against human CD93 protein.

Further, the nucleotide sequence of the light chain variable region of the rabbit monoclonal antibody is shown as SEQ ID NO.11, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 12.

In a third aspect the invention provides a recombinant vector or host cell comprising a nucleic acid molecule as described above.

In a fourth aspect, the invention provides the use of a rabbit monoclonal antibody against human CD93 protein as described above, a nucleic acid molecule as described above, a recombinant vector or host cell as described above in the preparation of a human CD93 protein immunoassay kit.

In a fifth aspect, the invention provides a human CD93 protein immunoassay kit comprising a rabbit monoclonal antibody directed against human CD93 protein as described above or an immunoconjugate of said rabbit monoclonal antibody conjugated to a detectable label.

Further, the kit is an enzyme-linked immunosorbent kit or a flow cytometry kit.

Further, the immunodetection samples include naturally expressed CD93 protein and recombinantly expressed CD93 protein in plasma, serum, cellular and tissue samples, wherein the cells include human histiocyte lymphoma cells or human multiple myeloma peripheral blood B lymphocytes.

The invention has the advantages and positive effects that:

The anti-CD 93 rabbit monoclonal antibody prepared by taking the extracellular region of the CD93 protein as an immunogen can specifically identify the recombinant expressed CD93 protein and the cell expressed natural CD93 protein, and the antibody has high affinity, good specificity and strong anti-interference capability for identifying and combining the human CD93 protein, can avoid false positive or false negative results caused by nonspecific combination during immunodetection, is beneficial to improving the accuracy and reliability of immunodetection, especially flow cell analysis results, and is suitable for detecting the CD93 protein with high specificity and high sensitivity.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing the results of detection of serum titers of New Zealand white rabbits immunized with CD93 protein by an ELISA method in example 1;

FIG. 2 is a graph showing the results of flow cytometry analysis and detection of the binding of immune serum of New Zealand white rabbits to positive cells U937, according to example 1, from left to right, with serum dilutions of 1:500 and 1:2000;

FIG. 3 is a graph showing the results of flow cytometry analysis and detection of the binding of immune serum of New Zealand white rabbits to negative cells Jurkat, according to example 1, from left to right, with serum dilutions of 1:500 and 1:2000;

FIG. 4 is a diagram of a mammalian vector pBR322 used in constructing a rabbit monoclonal antibody expression vector against human CD93 protein according to example 1 of the present invention, from left to right, a pRB322 vector map carrying a light chain constant region and a heavy chain constant region, respectively;

FIG. 5 is a graph showing the results of flow cytometry analysis and detection of the binding of the rabbit monoclonal antibody against human CD93 protein of example 2 of the present invention to positive cells U937, fluorescent markers 488；

FIG. 6 is a graph showing the results of flow cytometry analysis and detection of binding of a rabbit monoclonal antibody against human CD93 protein of example 2 of the present invention to a negative cell Jurkat, fluorescent markers488；

FIG. 7 is a graph showing the results of flow cytometry analysis and detection of the binding of the rabbit monoclonal antibody against human CD93 protein of example 2 of the present invention to positive cells U937, fluorescent markers647；

FIG. 8 is a graph showing the results of flow cytometry analysis and detection of binding of a rabbit monoclonal antibody against human CD93 protein of example 2 of the present invention to a negative cell Jurkat, fluorescent markers647。

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples. The examples described herein are intended to illustrate the invention only and are not intended to limit the invention.

Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit or scope of the appended claims. It is to be understood that the scope of the invention is not limited to the defined processes, properties or components, as these embodiments, as well as other descriptions, are merely illustrative of specific aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be within the scope of the following claims.

For a better understanding of the present invention, and not to limit its scope, all numbers expressing quantities, percentages and other values used in the present invention are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

In addition, it is noted that unless otherwise defined, in the context of the present invention, scientific and technical terms used should have meanings commonly understood by one of ordinary skill in the art.

The terms "comprising," "including," "having," and the like are intended to be non-limiting, as other steps and other ingredients not affecting the result may be added. The term "and/or" should be taken to refer to a specific disclosure of each of the two specified features or components with or without the other. For example, "a and/or B" will be considered to encompass the following: (i) A, (ii) B, and (iii) A and B.

In the context of the present invention, the terms "rabbit monoclonal antibody", "antibody" and the like have the same meaning and are used interchangeably to refer to antibodies that specifically bind to Human (Human) melanoma cell adhesion molecules (CD 93 protein). The modifier "rabbit" means that the complementarity determining regions (complementarity determining region, CDRs) of the antibody are derived from a rabbit immunoglobulin sequence.

An antibody is an immunoglobulin molecule capable of specifically binding to an antigen or epitope of interest through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In the present invention, the term "antibody" is to be interpreted in the broadest sense and includes different antibody structures, including but not limited to so-called full length antibodies, antibody fragments, and genetic or chemical modifications thereof, as long as they exhibit the desired antigen binding activity. Where "antibody fragment" refers to one or more portions or fragments of a full-length antibody, in typical examples, the antibody fragment comprises: fab, fab', F (ab) ₂、F(ab')₂、Fv、(Fv)₂、scFv、sc(Fv)₂.

A typical antibody molecule (full length antibody) consists of two identical light chains (L) and two identical heavy chains (H). Light chains can be divided into two types, kappa and lambda chains, respectively; heavy chains can be categorized into five, μ, δ, γ, α and ε chains, respectively, and antibodies are defined as IgM, igD, igG, igA and IgE, respectively. The amino acid sequences of the heavy and light chains near the N-terminus vary greatly, the other portions of the amino acid sequences are relatively constant, the region of the light and heavy chains near the N-terminus, where the amino acid sequences vary greatly, is referred to as the variable region (V), and the region near the C-terminus, where the amino acid sequences are relatively stable, is referred to as the constant region (C). Heavy chain variable regions (VH) and light chain variable regions (VL) are typically the most variable parts of antibodies and contain antigen recognition sites. The VH and VL regions can be further subdivided into hypervariable regions (hypervariable region, HVR) also known as Complementarity Determining Regions (CDRs) which are circular structures, and Framework Regions (FR) where the heavy and light chain CDRs are held closely together and cooperate with one another by the FR regions to form surfaces complementary to the three-dimensional structure of the antigen or epitope of interest, determining the specificity of the antibody, and are the sites for antibody recognition and binding to the antigen. The FR region is the more conserved part of VH and VL, which are generally in the β -sheet configuration, joined by three CDRs forming a connecting loop. Each VH and VL is typically composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

CDRs may be identified according to Kabat definitions, chothia definitions, a combination of both Kabat and Chothia definitions, abM definitions, contact definitions, IMGT unique numbering definitions and/or conformational definitions, or any CDR determination method known in the art. As used herein, CDRs are defined by the Kabat numbering system.

The light chain constant region (CL) and the heavy chain constant region (CH) are not directly involved in binding of an antibody to an antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of an antibody. CL lengths of different classes of igs (κ or λ) are substantially identical, but CH lengths of different classes of igs are different, e.g. IgG, igA and IgD include CH1, CH2 and CH3, while IgM and IgE include CH1, CH2, CH3 and CH4. The amino acid sequences of the antibody heavy and light chain constant regions are well known in the art.

Full length antibodies are the most complete antibody molecular structure, having a typical Y-type molecular structure, and thus, "full length antibodies", "complete antibodies" and "Y-type antibodies" are used interchangeably in the context of the present invention.

The term "Antigen binding fragment (Fab)" is a region of an antibody molecule that binds Antigen and consists of the complete light chain (variable and constant regions) and part of the heavy chain (variable and one constant region fragment), and fragments such as Fab, F (ab ') ₂, fab' can be obtained by proteolytic cleavage of the full-length antibody. For example, igG can be degraded into two Fab fragments and one Fc fragment by papain; igG can be degraded into a F (ab ') ₂ fragment and a pFC' fragment by pepsin. The F (ab ') ₂ fragment was further reduced to form two Fab' fragments. Because the Fab has an antigen binding region and a partial constant region, the Fab not only has antibody-antigen affinity like a single chain antibody (scFv), excellent tissue penetrating power and the like, but also has a more stable structure, and is widely applied to clinical diagnosis and treatment.

The term "variable fragment (Fv)" is located at the N-terminus of an antibody Fab fragment, contains only the variable region, and consists of one variable region of one light chain and one heavy chain, is a dimer of one VH and one VL that are non-covalently bound (VH-VL dimer), and the 3 CDRs of each variable region interact to form an antigen-binding site on the surface of the VH-VL dimer, with the ability to recognize and bind antigen, although with less avidity than an intact antibody.

The term "Single-chain antibody (scFv)" refers to a minimum antibody fragment in which a heavy chain variable region (VH) and a light chain variable region (VL) are linked by a flexible linker (linker, typically consisting of 10 to 25 amino acids), which retains the binding specificity of the original antibody to an antigen, and the linker in the present invention is not particularly limited as long as it does not interfere with the expression of the antibody variable regions linked at both ends thereof. Compared with full-length antibodies, scFv has the characteristic of small molecular weight, thus having higher penetrability and lower immune side reaction.

The full length sequences of the antibodies or antibody fragments of the invention may be from a single species, such as rabbit, or may be chimeric or humanized antibodies to reduce body rejection while maintaining the desired specificity, affinity. The term "chimeric antibody" antibody refers to an antibody in which a portion is derived from a particular source or species, while the remainder is derived from a different source or species. The term "humanized antibody" is a chimeric antibody in which the CDR regions of a non-human antibody, such as a rabbit antibody, and the FR regions derived from a human, in some cases, the variable regions of a non-human antibody bind to the constant regions of a human antibody, e.g., a human rabbit chimeric antibody; in other cases, the CDR regions of a non-human antibody bind to FR regions and constant regions derived from human antibody sequences, i.e., the CDR regions of a non-human antibody are grafted onto human antibody Framework (FR) sequences derived from single or multiple other human antibody variable region framework sequences. In the present invention, the CDR regions in the chimeric or humanized antibody are derived from rabbit-derived CDR regions.

The term "monoclonal antibody" refers to a homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen or epitope. "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies and is not to be construed as limiting the structure, source, or manner of preparation of the antibody. In some embodiments, the monoclonal antibodies are prepared by a hybridoma method, phage display method, yeast display method, recombinant DNA method, single cell screening, or single cell sequencing method.

The term "specific binding" is a term well known in the art that exhibits "specific binding," "specific binding," or is referred to as "preferential binding" if a molecule reacts more frequently, more rapidly, longer in duration, and/or with greater affinity to a particular antigen or epitope of interest than to other antigens or epitopes of interest, and does not necessarily require (although may include) exclusive binding.

In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.

The embodiment of the invention provides a rabbit monoclonal antibody for resisting human CD93 protein, which comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region comprise 3 Complementarity Determining Regions (CDRs), and the amino acid sequences of the complementarity determining regions 1,2 and 3 on the light chain variable region are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5; the amino acid sequences of the complementarity determining regions 1,2 and 3 on the heavy chain variable region are shown in SEQ ID NO.8, 9 and 10, respectively.

The invention selects the extracellular region of CD93 protein as immunogen to immunize white rabbits, then obtains the rabbit monoclonal antibody of the anti-CD 93 protein based on single B lymphocyte sorting and enrichment technology, and has high detection sensitivity on human CD93 protein when the obtained antibody is used for detection by an enzyme-linked immunosorbent assay; when the antibody is used for detecting by a flow cytometry, the specific fluorescent signal transition exists only in positive cells expressing the CD93 protein, and the fluorescent signal transition does not exist in negative cells not expressing the CD93 protein, so that the antibody can specifically identify the recombinant expressed CD93 protein and the natural CD93 protein expressed by the cells, and the antibody has high affinity, good specificity and strong anti-interference capability for identifying and combining with human CD93 protein, can avoid false positive or false negative results caused by nonspecific combination during immune detection, is beneficial to improving the accuracy and reliability of immune detection, especially the analysis result of the flow cytometry, and is suitable for detecting the CD93 protein with high specificity and high sensitivity.

Alternatively, the light chain variable region and the heavy chain variable region each comprise 4 Framework Regions (FR), 4 FR and 3 CDRs sequentially staggered to form the variable region. The amino acid sequence of the light chain variable region (VL) of the rabbit monoclonal antibody is shown as SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region (VH) is shown as SEQ ID NO. 7.

Optionally, the rabbit monoclonal antibodies of the invention further comprise a light chain constant region (CH) and a heavy chain constant region (VH), CL and VL comprising the complete light chain, CH and VH comprising the complete heavy chain. The constant regions of antibodies are typically obtained by public interrogation, such as: the rabbit source IGG GAMMA C REIGN was searched for CH and the rabbit source IGG KAPPA C REIGN was searched for CL via IMGT online database (www.imgt.org).

Specifically, the amino acid sequence of the light chain comprising CL is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain comprising CH is shown as SEQ ID NO. 6.

The rabbit monoclonal antibody of the invention may be a full length antibody or an antibody fragment, which refers to a polypeptide that retains substantially the same biological function or activity as the full length form of the rabbit monoclonal antibody, in particular, an antibody fragment comprising the CDR regions (SEQ ID No.3-5 and SEQ ID No. 8-10) as described above, more preferably having the variable regions (SEQ ID No.2 and SEQ ID No. 7) as described above, thereby retaining intact antigen recognition and binding sites capable of binding to the same antigen, in particular to the same epitope, as the full length antibody. Such antibody fragments include, but are not limited to: (i) A Fab fragment, a monovalent fragment consisting of the variable region and the first constant region of each heavy and light chain; (ii) A F (ab) ₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fv fragment consisting of one heavy chain variable region and one light chain variable region of an antibody; (iv) (Fv) ₂ fragment consisting of two Fv fragments covalently linked together; (v) An scFv fragment, an Fv fragment consisting of a single polypeptide chain, a heavy chain variable region and a light chain variable region joined by a linker; (vi) The sc (Fv) ₂ fragment is obtained by ligating two heavy chain variable regions and two light chain variable regions via a linker or the like. These antibody fragments can be obtained by conventional techniques known in the art.

Yet another embodiment of the invention provides a nucleic acid molecule encoding a rabbit monoclonal antibody as described above against human CD93 protein.

The nucleic acid molecule may be in the form of DNA (e.g., cDNA or genomic DNA or synthetic DNA) or RNA (e.g., mRNA or synthetic RNA). The DNA may be single-stranded or double-stranded, or may be a coding strand or a non-coding strand. The sequence of the nucleic acid molecule is deduced by conventional means such as codon encoding rules according to the amino acid sequence of the antibody.

Illustratively, the nucleotide sequence of the light chain variable region of the rabbit monoclonal antibody of the invention is shown as SEQ ID NO.11, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 12. It will be appreciated by those skilled in the art that nucleic acid molecules other than those exemplified above may likewise be encoded to provide rabbit monoclonal antibodies of the invention due to the degeneracy of the genetic code, and therefore the nucleic acid molecules of the examples should not be taken as limiting the scope of the invention.

The full-length sequence of the nucleic acid molecule or a fragment thereof can be obtained by PCR amplification, recombinant methods or artificial synthesis.

Another embodiment of the invention provides a recombinant vector or host cell comprising a nucleic acid molecule as described above.

The starting vector from which the recombinant vector is constructed is a variety of vectors conventional in the art, as long as it is capable of harboring the nucleic acid molecule. Typical vectors include plasmids, viral vectors, phages, cosmids and minichromosomes. Plasmids are the most common form of vector, and thus, in the context of the present invention, vectors are used interchangeably with plasmids. The vector may be a cloning vector (i.e., for transferring the nucleic acid molecule into a host and for mass propagation in a host cell) or an expression vector (i.e., comprising the necessary genetic elements to allow expression of the nucleic acid molecule inserted into the vector in a host cell). The cloning vector may contain a selectable marker and an origin of replication that matches the cell type specified by the cloning vector, while the expression vector contains regulatory elements (e.g., promoters, enhancers) for expression in the specified host cell. The nucleic acid molecules of the invention may be inserted into a suitable vector to form a cloning vector or an expression vector carrying the nucleic acid molecule. This is well known in the art and will not be described in detail herein.

Nucleic acid molecules encoding the heavy and light chains of the antibodies of the invention may be constructed separately on two vectors, which may be introduced into the same or different host cells. When the heavy and light chains are expressed in different host cells, each chain may be isolated from the host cell in which it is expressed and the isolated heavy and light chains mixed and incubated under appropriate conditions to form the antibody. In other embodiments, nucleic acid molecules encoding the heavy and light chains of a rabbit monoclonal antibody of the invention may also be cloned into a vector, each nucleic acid sequence being linked downstream of a suitable promoter; for example, each nucleic acid sequence encoding a heavy chain and a light chain may be operably linked to a different promoter, or the nucleic acid sequences encoding the heavy chain and the light chain may be operably linked to a single promoter such that both the heavy chain and the light chain are expressed from the same promoter. The choice of expression vector/promoter depends on the type of host cell used to produce the antibody.

Transformation of host cells with recombinant vectors can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which are capable of absorbing DNA, can be obtained after the exponential growth phase and treated with CaCl ₂ or MgCl ₂. Microinjection, electroporation, or liposome encapsulation may also be used if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, microinjection, electroporation, liposome packaging, and the like.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: bacterial cells of E.coli, streptomyces, salmonella typhimurium, fungal cells such as insect cells of yeast, drosophila S2 or Sf9, CHO, COS7, 293 series cells, etc. After obtaining a host cell transformed with the expression vector as described above, the cell is cultured under appropriate conditions to express the monoclonal antibody, and then isolated to obtain a purified antibody.

In a preferred embodiment, the recombinant vector is a mammalian expression vector pBR322 and the host cell is a human kidney epithelial cell (293F cell).

The embodiment of the invention also provides an application of the rabbit monoclonal antibody of the anti-human CD93 protein, the nucleic acid molecule, the recombinant vector or the host cell in preparation of a human CD93 protein immunoassay kit.

The application advantages of the anti-human CD93 protein rabbit monoclonal antibody, nucleic acid molecule, recombinant vector or host cell in preparing the human CD93 protein immunoassay kit are the same as those of the anti-human CD93 protein rabbit monoclonal antibody described above relative to the prior art, and are not described in detail herein.

Based on the same inventive concept, the embodiment of the invention also provides a human CD93 protein immunodetection kit, which comprises the rabbit monoclonal antibody aiming at the human CD93 protein or an immunoconjugate of the rabbit monoclonal antibody and a detectable label.

In the case of immunoassay, a sample to be examined is contacted with an anti-human CD93 protein monoclonal antibody, and then the monoclonal antibody is detected. In some embodiments, monoclonal antibodies directed against human CD93 protein may be conjugated to a detectable label to effect qualitative or quantitative detection of CD93 protein by analyzing the change in a recognizable signal produced by the detectable label. In other embodiments, the anti-human CD93 protein monoclonal antibody (primary or capture antibody) is not labeled, and the detectable label is conjugated to a secondary antibody (detection antibody) or other molecule that can bind to the monoclonal antibody. For example, if the anti-human CD93 protein monoclonal antibody is a rabbit IgG antibody, the secondary antibody may be an anti-rabbit IgG antibody, whereby the secondary antibody conjugated with a detectable label specifically binds to the primary antibody to produce a change in the recognizable signal.

Such detectable labels for producing identifiable signal changes include, but are not limited to: biotin, fluorescein, chemiluminescent groups, fluorescent proteins, enzymes (e.g., horseradish peroxidase, acid phosphatase), colloidal gold, colored magnetic beads, latex particles, radionuclides, detection antibodies, or combinations thereof.

In a preferred embodiment, the immunoassay method is an enzyme-linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA) or Flow Cytometry (FC), etc., and therefore, the kit is an enzyme-linked immunosorbent kit or a Flow cell kit.

Alternatively, the immunodetection samples include naturally expressed CD93 protein as well as recombinantly expressed CD93 protein in plasma, serum, cellular and tissue samples. Among them, cells include, but are not limited to: human histiocyte lymphoma cells, human multiple myeloma peripheral blood B lymphocytes.

The invention will be further illustrated with reference to specific examples. The experimental methods in which specific conditions are not specified in the following examples are generally conducted under conventional conditions, for example, those described in the molecular cloning Experimental guidelines (fourth edition) published in Cold spring harbor laboratory, or are generally conducted under conditions recommended by the manufacturer.

EXAMPLE 1 preparation of Rabbit monoclonal antibodies against human CD93 protein

1. Antigen preparation

The immunogen is a mammalian expression system, and the human recombinant CD93 protein 24-580aa is obtained by expression and purification, the full-length amino acid sequence of the CD93 protein is shown in Uniprot number O00274 and NCBI accession number NP-036204.2, and the gene sequence is shown in NCBI accession number NM-012072.4. CD93 protein 24-580aa is located in the extracellular region of CD93, the extracellular region detection is simpler and more convenient than that of the intracellular region, the background signal is lower, the gene sequence corresponding to the polypeptide segment is constructed into pYURK-RaFC vector, the high-quality recombinant Human CD93 mature protein with biological activity is expressed in 293F cells, and the purity of the obtained protein is more than 95%.

2. Immunization of animals

The human recombinant CD93 protein 24-580aa prepared above is used as immunogen to immunize 4 New Zealand white rabbits, each white rabbit is immunized by 200 mug, the immunogen is mixed with an equivalent amount of complete Freund's adjuvant (purchased from Sigma company) to prepare an emulsifier before the first immunization, and the emulsifier is subcutaneously injected at the abdomen and back of the rabbits. 100 μg of immunogen was mixed with an equal amount of incomplete Freund's adjuvant (purchased from Sigma company) every 3 weeks after the first immunization to prepare an emulsifier, which was subcutaneously injected at the abdomen and back of rabbits at multiple points to boost the immunization twice. Serum was taken after three immunizations according to 1: titers were measured by ELISA after 243000 dilution, rabbits with OD _450nm greater than 0.2 were boosted by subcutaneous multipoint injection with 200 μg immunogen once, spleens were taken three to four days later, and serum was diluted for final immune serum and assayed for affinity and specificity reactions with FC to the test cell samples.

The enzyme-linked immunosorbent assay (ELISA) method for immune serum titer detection comprises the following steps: 1) Coating: human recombinant CD93 protein 24-580aa (final concentration 1. Mu.g/mL) diluted with 1 XPBS buffer, coating solution was added at 25. Mu.L/well in 384 well plates, after balancing 384 well plates, plates were briefly centrifuged (stopped after reaching 1000 rpm) and overnight at 4 ℃; 2) Closing: the overnight plates at 4℃were removed and washed 5 times with 75. Mu.L of wash buffer (PBS containing 0.05% (v/v) Tween-20) added to each well with a plate washer; 50. Mu.L/well of blocking buffer (PBS containing 1% BSA, 0.5% gelatin and 5% sucrose) was added to the plates using a row gun and incubated for 1h at room temperature for blocking irrelevant binding sites; 3) Gradient dilution and sample adding of serum to be tested: the plate wash procedure of 2) was repeated, the serum to be tested was diluted in a gradient in 96-well plates and diluted with dilution buffer (PBS containing 1% bsa) at 1:1000, carrying out triple dilution, namely 8 gradients, wherein the dilution ratio can be adjusted according to actual conditions; 4) Secondary antibody incubation: the plate washing process of 2) is repeated, the plate is washed, and horseradish peroxidase (HRP) -coupled goat anti-rabbit IgG is diluted by a dilution buffer solution, wherein the dilution ratio is 1:5000, adding diluted goat anti-rabbit IgG-HRP into the plate at 25 μl/well by using a row gun for binding the target protein in the plate, and incubating for 1h at room temperature in the absence of light; 5) Termination reaction and color development: the plate washing process is repeated to wash the pore plate, TMB is added according to 100 mu L/pore for color development, light is prevented for 10min at 37 ℃, 100 mu L/pore of 0.5M oxalic acid solution is added for stopping the reaction, the absorption value of 450nm is measured, the rabbit serum before immunization is used as negative control, and the titer of the immune serum is judged by taking the ratio of the measured value to the control value as positive, wherein the ratio is more than or equal to 2.1.

The serum titer test results are shown in fig. 1, and it can be seen that the rabbit generates stronger immune response in the third immunization and the fourth boost immunization, the antibody titer is high, and the serum titer of the fourth immunization is optimal, and the method can be used for separating antibodies.

Flow Cytometric (FC) analysis of immune serum and cell samples includes the steps of: 1) Experiment preparation: ultraviolet irradiation sterilization super clean bench for 15-20min, and blower fan for 5min, preparing for aseptic work; 2) The dyeing method comprises the following steps: a) Collecting and washing cells, and then determining the total number of cells, and checking whether the cell viability is about 95% and not less than 90%; cells were resuspended to about 3X 10 ⁶-5×10⁶ cells/mL with 0.5% BSA/PBS and dispensed into 96-well V-plates at 50. Mu.L per well; b) The immune serum (used as primary antibody) diluted by PBS solution containing 0.5% BSA (0.5% BSA/PBS) is distributed into a 96-well V-shaped plate, cells in each well are resuspended, mixed and oscillated, and the reaction is carried out for 20min at room temperature; c) 400g was centrifuged for 5min, the supernatant was discarded, and washed 2 times with 200. Mu.L of 0.5% BSA/PBS; d) mu.L per well will be 1 with 0.5% BSA/PBS: 200 diluted Fluorescein (FITC) AffiniPure F (ab') 2Fragment Goat Anti-Rabbit IgG (purchased from jackson, cat# 111-096-046) was dispensed into 96-well V-plates, cells in each well resuspended, and mixed; e) Wrapping with aluminum foil paper, and gently mixing on a microplate vibration mixer for 20min; f) Washing 2 times with 200. Mu.L of 0.5% BSA/PBS, and finally re-suspending each well of cells with 200. Mu.L of 0.5% BSA/PBS, and preserving in dark; 3) Flow analysis: analysis was performed using AND maintaining SOP-105-AND-CA-008 operations with Beckman cytoflex flow analyzers. Rabbit IgG isotype control (from ABclonal, cat# AC 042) was used as isotype control.

The cell samples to be tested in this example include human histiocyte lymphoma cell U937 and human T lymphocyte leukemia cell Jurkat, and the FC detection results of immune serum and positive cell U937 expressing CD93 protein and negative cell Jurkat not expressing CD93 are shown in FIGS. 2-3 respectively: serum dilutions were 1 from left to right: 500. 1:2000, the abscissa of the graph shows the relative fluorescence intensity, the ordinate shows the relative cell number, the red curve is a negative control, the blue curve is a isotype control, and the yellow curve is the immune serum to be tested. As can be seen from the figure, the immune serum has specific binding in positive cells, better fluorescence transition, no non-specific binding in negative cells and no fluorescence transition compared with rabbit isotype control, and the immune serum can enter the stage of separating spleen cells after determining that high-specificity and high-affinity antibodies which can be used for flow detection are generated in animal bodies.

3. Isolation and culture of B lymphocytes in spleen

Isolation of spleen cells: taking out a culture dish in a safe cabinet in a sterile operation mode, adding 30-40mL of basic culture Medium (RPMI Medium 1640basic+1% Pen Strep; RPMI 1640 is purchased from Gibco, product number C11875500BT; pen Strep is purchased from Gibco, product number 15140-163), placing a cell screen, taking out the spleen, placing the spleen in the cell screen, cutting off superfluous connective tissues and fat on rabbit spleen tissues, cutting the spleen tissues, placing the spleen tissues in the cell screen for grinding, taking a clean grinding rod, grinding the tissues by rolling at the tail end of the pressed part of the grinding rod, and grinding thoroughly until the whole spleen tissues are nearly white as far as possible; the cells in the membrane slowly come out, are filtered by a cell screen into a basic culture medium, the cell screen is washed by 10mL of the basic culture medium, the basic culture medium outside the cell screen is collected, the cell screen is centrifuged for 5min at room temperature by using 400g of centrifugal force, the supernatant is sucked and removed, the cells are reserved, and 13mL of RBC erythrocyte lysate (BD PHARM LYSE 555899) at normal temperature is added; after the cells are gently blown by a pipetting gun for several times, timing for 1min, performing erythrocyte lysis, adding 37mL of basic culture medium to terminate the reaction after timing is completed, centrifuging for 5min at room temperature by using a centrifugal force of 400g, sucking and discarding the supernatant, and reserving the cells; adding 40mL of basal medium placed at normal temperature, blowing and beating the resuspended cells by a pipetting gun, centrifuging for 5min at room temperature by using a centrifugal force of 400g, sucking and discarding the supernatant, and reserving the cells; 20mL of a B cell culture medium placed at room temperature was added, the cell mass was gently blown off with a pipette, the cells were resuspended, and the cells were filtered again through a screen, after which the cells were counted.

Sorting and culturing of B lymphocytes: see patent "method for efficiently separating single antigen-specific B lymphocytes from spleen cells (publication No. CN110016462A, publication No. 2019-07-16)" and patent "an in vitro culture system for B lymphocytes and application (publication No. CN111518765A, publication No. 2020-08-11)".

4. Cloning of genes encoding Rabbit monoclonal antibodies

B lymphocyte positive clones capable of recognizing and binding to human CD93 protein were detected by antigen-coated ELISA, the cells were collected, RNA was extracted by Quick-RNA ^TM Micro Prep kit (purchased from ZYMO Co., ltd., cat. No. R1100-250) after lysis, and reverse transcribed into cDNA. The cDNA is used as a template, a PCR method is adopted to amplify the light chain variable region (VL) and heavy chain variable region (VH) genes of a naturally paired rabbit monoclonal antibody from the cDNA of the corresponding positive clone, and a plurality of clones are selected for sequencing, and the sequencing work is completed by Jin Kairui biotechnology company. The PCR reaction system comprises: 4. Mu.L of cDNA, 1. Mu.L of forward primer (10 mM), 1. Mu.L of reverse primer (10 mM), 12.5. Mu.L of 2X Gloria HiFi (from Wuhan Aibotac, cat. RK 20717) and 6.5. Mu. L H ₂ O. The PCR amplification procedure included: the reaction mixture was subjected to preliminary denaturation at 98℃for 30s, followed by 40 cycles at 98℃for 10s,64℃for 30s, and 72℃for 30s, and finally kept at 72℃for 5min, and the resulting reaction mixture was kept at 4 ℃.

The nucleic acid sequences (5 '-3') of the forward primer (F) and the reverse primer (R) for amplifying the VL and VH genes are shown below:

VL-F: TGAATTCGAGCTCGGTACCCATGGACACGAGGGCCCCCAC (see SEQ ID NO. 13);

VL-R: CACACACACGATGGTGACTGTTCCAGTTGCCACCTGATCAG (see SEQ ID NO. 14);

VH-F: TGAATTCGAGCTCGGTACCCATGGAGACTGGGCTGCGCTG (see SEQ ID NO. 15);

VH-R: GTAGCCTTTGACCAGGCAGCCCAGGGTCACCGTGGAGCTG (see SEQ ID NO. 16)

5. Production and purification of Rabbit monoclonal antibodies

To obtain a plurality of rabbit monoclonal antibodies recognizing human CD93 protein, the light chain variable region (VL) and heavy chain variable region (VH) genes of the selected rabbit monoclonal antibodies are inserted into mammalian expression vectors pBR322 carrying light chain constant region (CL) and heavy chain constant region (CH) genes, respectively, to form expression vectors carrying antibody heavy chain and light chain genes. Wherein, the expression pattern of the mammalian expression vector pBR322 is shown in FIG. 4: in the figure, pRB322 origin and f1 origin are replication promoters in E.coli (E.Coli), AMPCILLIN is a plasmid resistance gene, CMV IMMEARLY promotor is a promoter in eukaryotes, SV40 PA terminator is a tailing signal, LIGHT CHAIN constant is a nucleic acid sequence of a light chain constant region (left panel), HEAVY CHAIN constant is a nucleic acid sequence of a heavy chain constant region (right panel), and constant region sequences are obtained by querying an IMGT online database (www.imgt.org). The specific construction process is as follows: mammalian cell expression vectors containing the CL and CH genes of the rabbit monoclonal antibodies were routinely linearized with XbaI and NheI restriction enzymes, respectively. And then purifying the amplified PCR products (the VH gene and the VL gene with signal peptides at the upstream), respectively constructing heavy chain variable region genes and light chain variable region genes into corresponding mammal expression vectors by adopting a homologous recombination mode, and carrying out sequencing verification.

The signal peptide of this example may be expressed by using an antibody commonly used in the art, such as the "high affinity Human IL-5 rabbit monoclonal antibody and its use (publication No. CN115819578A, publication No. 2023-03-21)", the light chain variable region has the signal peptide "MDTRAPTQLLGLLLLWLPGARC" upstream and the heavy chain variable region has the signal peptide "METGLRWLLLVAVLKGVQC" upstream.

The successfully constructed expression vector containing the light chain gene and the heavy chain gene of the corresponding rabbit monoclonal antibody is transfected into 293F cells together for 72 to 96 hours to obtain the recombinant rabbit monoclonal antibody which is used for recognizing human CD93 in the culture supernatant; recombinant rabbit monoclonal antibodies recognizing human CD93 were then purified from the transfected culture supernatant using protein A affinity gel resin (available from Tiandi and, cat. SA 023100), and the purification protocol was followed according to the protein A affinity gel resin instructions, which are not described here. Antibody purity was verified using 12% SDS-PAGE gel electrophoresis and the antibody concentration was calculated to be 1.0mg/mL with a purity of greater than 95%.

The rabbit monoclonal antibody against human CD93 protein obtained in this example was designated 1G8, and the Amino Acid (AA) and nucleic acid (DNA) sequences thereof are shown in Table 1, and for convenience of description, the light chain complementarity determining regions CDR1, CDR2 and CDR3 are denoted by LCDR1, LCDR2 and LCDR3, and the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 are denoted by HCDR1, HCDR2 and HCDR3, respectively.

TABLE 1 summary of sequence information of rabbit monoclonal antibody 1G8 of the invention

EXAMPLE 2 research on the use of Rabbit monoclonal antibody 1G8

Flow Cytometry (FC) of rabbit monoclonal antibody 1G 8: the cell samples of this example included positive cell-human histiocyte lymphoma cell U937 and negative cell-human T lymphocyte leukemia cell Jurkat at a cell concentration of 1E 6/100. Mu.L. The method of measurement is described in example1, except that the present example uses direct antibodies, i.e., rabbit monoclonal antibodies 1G8 are coupled to each other488 And the like647 Fluorescence is used as a detection antibody, a fluorescent secondary antibody is not required to be used,488 Rabbit anti-human CD93 antibody647 Rabbit anti-human CD93 antibody working concentration was 2 μg/mL.

FIGS. 5-8 show the results of flow cytometry detection of the binding of anti-human CD93 protein rabbit monoclonal antibody 1G8 to positive cell U937 and negative cell Jurkat, respectively, FIGS. 5-6 show the use of fluorescent signals for positive cell U937 and negative cell Jurkat, respectively488, FIGS. 7-8 show the fluorescence signal of positive cell U937 and negative cell Jurkat, respectively647, The abscissa of the above graph shows the relative fluorescence intensity, the ordinate shows the relative cell number, the red curve is a negative control, the blue curve is a isotype control, and the yellow curve is rabbit monoclonal antibody 1G8. As can be seen from the figure, the rabbit monoclonal antibody 1G8 has specific binding on positive cell U937 and no non-specific binding on negative cell Jurkat, which indicates that the rabbit monoclonal antibody 1G8 can specifically identify natural CD93 protein expressed in human cells, has high affinity for binding human CD93 protein,488 And the like647 Fluorescent transition signals are obvious and have no non-specific recognition, which shows that the antibody has high specificity and good anti-interference capability, and is beneficial to improving the accuracy and reliability of immune detection.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims

1. The rabbit monoclonal antibody for resisting human CD93 protein is characterized by comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of a complementarity determining region 1, a complementarity determining region 2 and a complementarity determining region 3 on the light chain variable region are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5; the amino acid sequences of the complementarity determining regions 1,2 and 3 on the heavy chain variable region are shown in SEQ ID NO.8, 9 and 10, respectively.

2. The rabbit monoclonal antibody against human CD93 protein according to claim 1, wherein the amino acid sequence of said light chain variable region is shown in SEQ ID No.2 and the amino acid sequence of said heavy chain variable region is shown in SEQ ID No. 7.

3. The rabbit monoclonal antibody against human CD93 protein of claim 2, further comprising a light chain constant region and a heavy chain constant region, wherein said light chain constant region and said light chain variable region comprise a light chain, and wherein said light chain has the amino acid sequence shown in SEQ ID No. 1; the heavy chain constant region and the heavy chain variable region form a heavy chain, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 6.

4. The rabbit monoclonal antibody against human CD93 protein according to claim 1 or 2, wherein said rabbit monoclonal antibody is a full-length antibody or an immunologically active antibody fragment; the antibody fragment is selected from at least one of Fab fragment, F (ab) ₂ fragment, fv fragment, (Fv) ₂ fragment, scFv fragment and sc (Fv) ₂ fragment.

5. The rabbit monoclonal antibody against human CD93 protein according to claim 1, wherein the immunogen for preparing said rabbit monoclonal antibody is CD93 protein 24-580aa.

6. A nucleic acid molecule encoding a rabbit monoclonal antibody against human CD93 protein according to any one of claims 1-4.

7. The nucleic acid molecule of claim 6, wherein the nucleotide sequence of the light chain variable region of the rabbit monoclonal antibody is shown in SEQ ID NO.11 and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO. 12.

8. A recombinant vector or host cell comprising the nucleic acid molecule of any one of claims 6-7.

9. A human CD93 protein immunoassay kit comprising the rabbit monoclonal antibody against human CD93 protein of any one of claims 1-5 or an immunoconjugate of said rabbit monoclonal antibody conjugated to a detectable label.

10. The human CD93 protein immunoassay kit of claim 9, wherein said kit is an enzyme-linked immunosorbent kit or a flow cell kit;

The immunodetection samples comprise CD93 protein naturally expressed in plasma, serum, cells and tissue samples and CD93 protein recombinantly expressed, wherein the cells comprise human histiocyte lymphoma cells or human multiple myeloma peripheral blood B lymphocytes.